T-cell receptor and nucleic acid encoding the receptor

ABSTRACT

A polypeptide comprising a polypeptide consisting of an amino acid sequence shown in SEQ ID NO: 5 of Sequence Listing or a polypeptide consisting of an amino acid sequence having deletion, addition, insertion or substitution of one to several amino acid residues in the sequence, the polypeptide being capable of constituting an HLA-A24-restricted, MAGE-A4 143-151 -specific T cell receptor together with a polypeptide consisting of an amino acid sequence shown in SEQ ID NO: 2 of Sequence Listing.

TECHNICAL FIELD

The present invention relates to a polypeptide constituting an α-chainof an HLA-A2402-restricted T cell receptor (TCR) specific toMAGE-A4₁₄₃₋₁₅₁, a nucleic acid encoding the polypeptide, a polypeptideconstituting a β-chain of the above-mentioned TCR, a nucleic acidencoding the polypeptide, a T cell receptor containing theabove-mentioned polypeptide constituting the α-chain and theabove-mentioned polypeptide constituting the β-chain, a recombinantnucleic acid containing the nucleic acid, a vector carrying therecombinant nucleic acid, a cell into which the above-mentioned nucleicacid or vector is transduced, and a carcinostatic agent containing as anactive ingredient the above-mentioned vector or cell.

BACKGROUND ART

Cytotoxic T cells (CTLs) include those capable of recognizing a complexby a specific T cell receptor (hereinafter simply referred to as “TCR”),the complex that is a conjugate of a major histocompatibility genecomplex (hereinafter simply referred to as “MHC”)-encoding majorhistocompatibility antigen molecule (MHC molecule; in a case of human,it is called “human leukocyte antigen,” hereinafter simply referred toas “HLA”) and an antigen peptide, and killing a cell in which thecomplex is presented on a cell surface. Therefore, in order to establishthe cytotoxic reaction, it is necessary that 1) a CTL having a TCRspecific to HLA Class I type of a target cell exists, and 2) an antigenpeptide so that a complex formed by binding to the HLA molecule iscapable of being recognized by the TCR exists.

The antigen peptide as described above is generated by, for example,processing an antigen or the like synthesized in a cell of a mammaliancell in a cytoplasm, thereby degrading into small peptides. The smallpeptides are further associated with an HLA molecule to be presented onthe cell surface. In other words, in the proteasome complex consistingof numerous subunits, a protein is degraded into peptides consisting of8 to 15 amino acids, some of which are transported from the cytoplasm tothe endoplasmic reticulum by a TAP transporter. Once these peptides canbe bound to a heterodimer of Class I/β2 microglobulin, they arestabilized in the form of a trimolecular complex, and transported to thecell surface through the Golgi apparatus. A tumor cell expressing atumor-associated antigen or tumor-specific antigenic protein issupposedly capable of presenting an HLA-restricted antigenic peptiderecognized by a T cell.

It has been known that the HLA Class I molecules are mainly HLA-A, -B,and -C, that the antigen peptides which are presented by binding tothese molecules consist of 8 to 10 amino acids, and further that thereare given different structural features for each of the HLA molecules.For example, as a peptide binding to an HLA-A2.1 molecule which is mostfrequently found worldwide, a peptide consisting of 9 to 10 amino acids,the peptide having Leu at a second position from an N-terminal, and Leuor Val at a C terminal has been most well known. In addition, as apeptide binding to an HLA-A24 molecule which is found more richly in theAsians such as the Japanese, a peptide consisting of 9 to 10 aminoacids, the peptide having any one of Tyr, Phe, Met, and Trp at a secondposition from an N-terminal, and any one of Leu, Ile, Trp, and Phe at aC-terminal has been most well known.

Tumor antigens for which antigen peptides have so far been identifiedare MAGE-A1, MAGE-A3, and MAGE-A4 against HLA-A1; MAGE-A3, MART1,tyrosinase, gp100, HER2/neu, CEA and the like against HLA-A2.1; MAGE-A3against HLA-Cw1; MAGE-A3 against HLA-B44; MAGE-A4 against HLA-B37; andMAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, NY-ESO-1, CEA, HER2/neu, tyrosinase,β-catenin, and the like against HLA-A24. In many of these tumorantigens, first, a cell line is established for a Class I-restricted CTLrecognizing a tumor cell, a tumor antigen recognized by the CTL isidentified, a minimum unit in tumor antigen proteins is subsequentlyfound by a genetic engineering method, and peptides in the minimum unitare further found on the basis of the information regarding the bindingmotif to the HLA Class I molecules. In addition, first, peptides bindingto the HLA Class I molecules are found in the tumor antigen proteins onthe basis of the motif structure commonly shared between theabove-mentioned peptides binding to the HLA Class I molecules, thosepeptides from which CTLs are inducible are subsequently selectedutilizing an antigen presenting cell, and finally antigen peptides arethen determined depending upon whether or not a CTL having toxicityagainst a tumor cell can be induced.

On the other hand, the HLA Class I molecules are classified into somesubtypes, and the kinds of owned subtypes greatly differ among races.Worldwide, HLA-A2 is most often found, and 45% of the Caucasians areHLA-A2-positive. Moreover, the identification of this HLA-A2-restrictedantigen peptide is most advanced. In the Japanese, HLA-A2-positive isfound in 40%, and when their subtypes are studied, HLA-A*0201-positive,which is the same as the Caucasians, is 20%, and much of the remainingare A*0206-positive. The binding peptides to these subtypes aredifferent, so that HLA-A2 that is mainly studied is HLA-A*0201. On theother hand, in the Japanese, HLA-A24-positive is found in 60% or more,and an HLA-A24-positive percentage is higher in the Asians than otherraces. Therefore, the finding of an HLA-A24-restricted antigen peptideplays an important role in providing a CTL that is useful in thetreatment of tumor by inducing a CTL acting specifically to a tumor cellin the Asians, especially the Japanese.

Since antigen peptides differ on the basis of the difference in the HLAeven with the same antigen, the induction of a CTL utilizing antigenpeptides is complicated. Although various contrivances have been made inorder to solve this disadvantage, a satisfactory fruit has not yet beenobtained. One contrivance is a method including the steps of transducingan antigenic gene into an antigen presenting cell derived from a patienthim/herself (self), and inducing a T cell utilizing the transformedgene. As the antigen presenting cell, B cell, macrophage, and dendriticcell have been studied, and a clinical test has been carried out usingas an adjuvant or the like for a vaccine centering about dendritic cellwhich is known as a professional antigen presenting cell. However, thereis a disadvantage that much labor is required in furnishing theseantigen presenting cells in an amount necessary for immune induction. Bcell can be mass-produced by immortalization by EB virus; however, thereis a disadvantage in safety from the viewpoint of use of a virus.

As tumor antigen-specific TCR genes, for example, genes such asHLA-A2-restricted, MART1-specific TCR [Non-Patent Publication 1],MAGE-A3-specific TCR [Non-Patent Publication 2], CAMEL (CTL-recognizedantigen on melanoma)-specific TCR [Non-Patent Publication 3],gp100-specific TCR [Non-Patent Publication 4], NY-ESO-1-specific TCR[Non-Patent Publication 5], HLA-24-restricted WT1 (Wilms tumor1)-specific TCR [Non-Patent Publication 6], HLA-Cw16-restrictedMAGE-A1-specific TCR [Non-Patent Publication 7] have been cloned.

It can be expected to give a specific cytotoxic activity to an intendedantigen by transducing TCR gene into a given CTL. Based on the above,gene therapies with TCR gene targeted to MART1[Non-Patent Publication8], gp100 [Non-Patent Publication 4] and mHAG HA-2 antigen [Non-PatentPublication 9] have been tried.

MAGE-A4 is an antigen belonging to a MAGE subfamily of a cancer-testisantigen family, which is expressed in various cancers and has a highantigenicity (positive in 60% of esophageal cancer, 50% of head and neckcancers, 24% of non-small cell pulmonary cancer, 33% of stomach cancer,and 21% of Hodgkin disease), so that the antigen is expected to serve asa target antigen in cancer vaccine therapy. An HLA-A24-restrictedMAGE-A4₁₄₃₋₁₅₁ peptide-specific CTL clone has been obtained [Non-PatentPublication 10].

-   Non-Patent Publication 1: Cancer Res., 54, 5265-5268 (1994)-   Non-Patent Publication 2: Anticancer Res., 20, 1793-1799 (2000)-   Non-Patent Publication 3: Int. J. Cancer, 99, 7-13 (2002)-   Non-Patent Publication 4: J. Immunol. 170, 2186-2194 (2003)-   Non-Patent Publication 5: J. Immunol., 174, 4415-4423 (2005)-   Non-Patent Publication 6: Blood, 106, 470-476 (2005)-   Non-Patent Publication 7: Int. Immunol., 8, 1463-1466 (1996)-   Non-Patent Publication 8: J. Immunol., 163, 507-513 (1999)-   Non-Patent Publication 9: Blood, 103, 3530-3540 (2003)-   Non-Patent Publication 10: Clin. Cancer Res., 11, 5581-5589 (2005)

DISCLOSURE OF INVENTION Problems to be Solved by the Invention

As an HLA-A24-restricted TCR gene against a tumor-associated antigen,those genes against WT1 have been known, but the analyses therefor havestill been delayed as compared to those of HLA-A2.1, thereby making itimpossible to provide TCR genes that are useful in the treatment oftumor for the Asians, especially the Japanese. Therefore, a finding ofnew, HLA-A24-restricted TCR genes against various tumor antigens hasbeen desired.

One that plays the role of cytotoxic activity via the TCR in the livingbody is a CTL having antigen-specific TCRs, and there are somedisadvantages in the handling of these cells, for example, collectingand expanding the cells, in order to use them for the treatment of adisease such as cancer by proliferating the CTLs ex vivo. Therefore, ithas been earnestly desired to provide TCR gene specific to a tumorantigen or the like, in order to massively and easily prepare CTLshaving a desired antigen-specificity.

Means to Solve the Problems

As a result of intensive studies on the CTL against the tumor antigen,the present inventors have succeeded in cloning cDNAs encoding α-chainand β-chain of the TCR from the HLA-A24-restricted CTL against a tumorantigen MAGE-A4. Further, a cell, such as a CTL, expressing an HLA-A24molecule shows cytotoxicity specific to a peptide derived fromHLA-A24-restricted MAGE-A4 by transducing RNA prepared from these cDNAsinto these cells, and the present invention has been accomplishedthereby.

A first embodiment of the present invention relates to a polypeptideconstituting an HLA-A24-restricted, MAGE-A4₁₄₃₋₁₅₁-specific T cellreceptor, and a polypeptide comprising a variable region polypeptide ofthe above-mentioned receptor.

A second embodiment of the present invention relates to anHLA-A24-restricted, MAGE-A4₁₄₃₋₁₅₁-specific TCR, characterized in thatthe TCR contains the polypeptide as defined in the first embodiment ofthe present invention.

A third embodiment of the present invention relates to a nucleic acidencoding an HLA-A24-restricted, MAGE-A4₁₄₃₋₁₅₁-specific TCR, and nucleicacid encoding a polypeptide comprising a variable region polypeptide ofthe above-mentioned receptor.

A fourth embodiment of the present invention relates to a recombinantnucleic acid containing the nucleic acid as defined in the thirdembodiment of the present invention.

A fifth embodiment of the present invention relates to a vector carryingthe recombinant nucleic acid as defined in the fourth embodiment of thepresent invention, wherein the recombinant nucleic acid is inserted intothe vector.

A sixth embodiment of the present invention relates to a cell whichexpresses an HLA-A24-restricted, MAGE-A4₁₄₃₋₁₅₁-specific TCR,characterized in that the nucleic acid as defined in the thirdembodiment of the present invention is transduced into the cell, or thecell is transformed with the vector as defined in the fifth embodiment.

A seventh embodiment of the present invention relates to a carcinostaticagent, characterized in that the carcinostatic agent contains as anactive ingredient the cell as defined in the sixth embodiment of thepresent invention or the vector as defined in the fifth embodiment.

An eighth embodiment of the present invention relates to a method oftreating cancer, including the step of administering the carcinostaticagent as defined in the seventh embodiment of the present invention.

Effects of the Invention

According to the present invention, a nucleic acid encoding an α-chainand β-chain of an HLA-A24-restricted, MAGE-A4₁₄₃₋₁₅₁-specific TCR isprovided. In addition, a method for damaging a tumor cell using anHLA-A24-non-restricted, or MAGE-A4₁₄₃₋₁₅₁-non-specified T cell as aneffector cell is provided. The above-mentioned effector cell is useful,for example, in the treatment of cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the office upon request and paymentof the necessary fee.

FIG. 1 Graphs showing the results of tetramer assay of #2-28 cells,RNA-transduced ms69 cells, and ms69 cells.

FIG. 2 Graphs showing the results of tetramer assay of #2-28 cells,RNA-transduced CD8 positive cells, and CD8 positive cells.

FIG. 3 A chart showing the results of ELISPOT assay of #2-28 cells,RNA-transduced ms69 cells, and ms69 cells.

FIG. 4 A chart showing the results of ELISPOT assay of RNA-transducedCD8 positive cells, and CD8 positive cells.

FIG. 5 Graphs showing cytotoxic activity of #2-28 cells, RNA-transducedms69 cells, and ms69 cells.

FIG. 6 Graphs showing cytotoxic activity of #2-28 cells, RNA-transducedCD8 positive cells, and CD8 positive cells.

BEST MODE FOR CARRYING OUT THE INVENTION

A first embodiment of the present invention relates to a polypeptideconstituting an HLA-A24-restricted, MAGE-A4₁₄₃₋₁₅₁-specific T cellreceptor, the polypeptide comprising a variable region polypeptide ofthe above-mentioned receptor. There are two kinds of the above-mentionedpolypeptides, TCR α-chain and TCR β-chain polypeptides, and both chainsare combined to constitute an HLA-A24-restricted,MAGE-A4₁₄₃₋₁₅₁-specific TCR.

The above-mentioned α-chain polypeptide is capable of forming anHLA-A24-restricted, MAGE-A4₁₄₃₋₁₅₁-specific TCR together with that of aβ-chain, and the phrase “α-chain polypeptide” means a polypeptidecomprising an α-chain variable region polypeptide selected from apolypeptide of the amino acid sequence shown in SEQ ID NO: 5 of SequenceListing, and a polypeptide having deletion, addition, insertion orsubstitution of one to several amino acid residues in the amino acidsequence shown in SEQ ID NO: 5 of Sequence Listing. The polypeptidederived from an α-chain of the TCR in the present invention contains asan essential constituent the above-mentioned amino acid sequence of anα-chain variable region, or a sequence similar thereto. A polypeptideconsisting of the amino acid sequence of the entire α-chain (SEQ IDNO: 1) or a sequence similar thereto, i.e. an amino acid sequence havingdeletion, addition, insertion or substitution of one to several aminoacid residues, the sequence containing a constant region, is one ofpreferred embodiments of the present invention.

In addition, the above-mentioned β-chain polypeptide is capable offorming an HLA-A24-restricted, MAGE-A4₁₄₃₋₁₅₁-specific TCR together withthat of an α-chain, and the phrase “β-chain polypeptide” means apolypeptide comprising a β-chain variable region polypeptide selectedfrom a polypeptide of the amino acid sequence shown in SEQ ID NO: 7 ofSequence Listing, and a polypeptide having deletion, addition, insertionor substitution of at least one amino acid residue in the amino acidsequence shown in SEQ ID NO: 7 of Sequence Listing. The polypeptidederived from a β-chain of the TCR in the present invention contains asan essential constituent the above-mentioned amino acid sequence of aβ-chain variable region, or a sequence similar thereto. A polypeptideconsisting of the amino acid sequence of the entire β-chain (SEQ ID NO:2) or a sequence similar thereto, i.e. an amino acid sequence havingdeletion, addition, insertion or substitution of one to several aminoacid residues, the sequence containing a constant region, is one ofpreferred embodiments of the present invention.

The phrase “HLA-A24-restricted, MAGE-A4₁₄₃₋₁₅₁-specific TCR” as usedherein refers to a receptor capable of specifically recognizing acomplex of a peptide comprising the amino acid sequence as shown in SEQID NO: 9 of Sequence Listing (MAGE-A4₁₄₃₋₁₅₁, hereinafter simplyreferred to as P143) and an HLA-A24 molecule, and giving a T cellHLA-A24-restricted P143-specific cytotoxic activity against the targetcell when the TCR exists on the surface of the T cell. A specificrecognition of the above complex may be confirmed by a known method, andpreferred methods include, for example, tetramer analysis using HLA-A24molecule and P143, and ELISPOT assay. By performing the ELISPOT assay,it can be confirmed that a T cell expressing the TCR on the cell surfacerecognizes a target cell by the TCR, and that the signal is transmittedintracellularly. The confirmation that the above-mentioned complex cangive a T cell cytotoxic activity when the complex exists on the T cellsurface may also be carried out by a known method. A preferred methodincludes, for example, the determination of cytotoxic activity againstan HLA-A24-positive target cell, such as chromium release assay.

The polypeptide of the present invention can be produced in a geneticengineering manner using the nucleic acid of the present inventiondescribed later. The HLA-A24-restricted, MAGE-A4₁₄₃₋₁₅₁-specific TCR isallowed to express in a cell by, for example, transducing both of thenucleic acid encoding an α-chain polypeptide and the nucleic acidencoding a β-chain polypeptide mentioned above into the cell, andallowing to express α-chain and β-chain polypeptides.

A second embodiment of the present invention relates to anHLA-A24-restricted, MAGE-A4₁₄₃₋₁₅₁-specific TCR, characterized in thatthe TCR comprises the polypeptide of the present invention. Theabove-mentioned TCR can be prepared in the form which is separated fromnaturally concomitant biological components by, for example,artificially expressing a polypeptide encoded by the nucleic acid of thepresent invention described later using the above-mentioned nucleicacid, without intending to particularly limit the present inventionthereto.

A third embodiment of the present invention relates to a nucleic acidencoding an HLA-A24-restricted, MAGE-A4₁₄₃₋₁₅₁-specific TCR or avariable region thereof.

The nucleic acid of the present invention refers to a nucleic acidencoding a polypeptide comprising a TCR α-chain variable regionpolypeptide or a nucleic acid encoding a polypeptide comprising a TCRβ-chain variable region polypeptide, in which in a case where each ofthe nucleic acids is transduced into a cell together with a nucleic acidencoding a TCR β-chain polypeptide or a nucleic acid encoding a TCRα-chain polypeptide, a molecule binding specifically to anHLA-A24-restricted MAGE-A4₁₄₃₋₁₅₁ complex is allowed to express in theabove-mentioned cell. Here, the nucleic acid encoding a polypeptidecomprising a TCR α-chain variable region polypeptide includes a nucleicacid encoding a TCR α-chain polypeptide and a nucleic acid encoding aTCR α-chain variable region polypeptide; and the nucleic acid encoding apolypeptide comprising a TCR β-chain variable region polypeptideincludes a nucleic acid encoding a TCR β-chain polypeptide and a nucleicacid encoding a TCR β-chain variable region polypeptide. TheHLA-A24-restricted, MAGE-A4₁₄₃₋₁₅₁-specific TCR is allowed to express inthe above-mentioned cell even in cases where any of the combinations ofa nucleic acid encoding a TCR α-chain polypeptide and a nucleic acidencoding a TCR β-chain polypeptide, or a nucleic acid encoding a TCRα-chain variable region polypeptide and a nucleic acid encoding a TCRβ-chain variable region polypeptide are transduced into the cell.

Although not intended to limit the present invention thereto, theabove-mentioned nucleic acid encoding the α-chain polypeptide isexemplified by a nucleic acid consisting of the nucleotide sequenceshown in SEQ ID NO: 3 of Sequence Listing, and a nucleic acid capable ofhybridizing to a nucleic acid of the above-mentioned nucleotide sequenceor a complementary strand thereto under stringent conditions. Thenucleic acid encoding a variable region of the α-chain polypeptide isexemplified by a nucleic acid consisting of the nucleotide sequenceshown in SEQ ID NO: 6 of Sequence Listing, and a nucleic acid capable ofhybridizing to a nucleic acid of the above-mentioned nucleotide sequenceor a complementary strand thereto under stringent conditions. Inaddition, the above-mentioned nucleic acid encoding the β-chainpolypeptide is exemplified by a nucleic acid consisting of thenucleotide sequence shown in SEQ ID NO: 4 of Sequence Listing, and anucleic acid capable of hybridizing to a nucleic acid of theabove-mentioned nucleotide sequence or a complementary strand theretounder stringent conditions. The nucleic acid encoding a variable regionof the β-chain polypeptide is exemplified by a nucleic acid consistingof the nucleotide sequence shown in SEQ ID NO: 8 of Sequence Listing,and a nucleic acid capable of hybridizing to a nucleic acid of theabove-mentioned nucleotide sequence or a complementary strand theretounder stringent conditions.

The term “stringent conditions” as referred to herein includes, forexample, those conditions described, for example, in Molecular Cloning:A Laboratory Manual, 2nd Ed. edited by J. Sambrook et al., published byCold Spring Harbor Laboratory, and the like. Concretely, the conditionsinclude, for example, conditions of incubating in a solution of 6×SSCcontaining 0.5% SDS, 5×Denhardt's solution and 0.01% denatured salmonsperm DNA at 65° C. for 12 to 20 hours together with a probe. Thenucleic acid hybridized to the probe can be detected after washing in asolution of 0.1×SSC containing 0.5% SDS at 37° C. to remove a probenon-specifically bound thereto.

The term “nucleic acid” as used herein means a single-stranded ordouble-stranded DNA or RNA, a DNA-RNA chimeric mixture, or a DNA-RNAhetero-double-strand. In a case where all or a part of the nucleic acidsare RNA, in the sequence of the RNA moiety, “T” can be read as “U” inSequence Listing described in the specification of the presentinvention. The preferred embodiment of the present invention isexemplified by combinations of two kinds of nucleic acids: a nucleicacid encoding a TCR α-chain polypeptide or a nucleic acid encoding a TCRα-chain variable region polypeptide, and a nucleic acid encoding a TCRβ-chain polypeptide or a nucleic acid encoding a TCR β-chain variableregion polypeptide, each in the present invention. The above-mentionedcombinations of the nucleic acids are useful for the purpose ofexpressing the HLA-A24-restricted, MAGE-A4₁₄₃₋₁₅₁-specific TCR in acell.

The nucleic acid of the present invention can be obtained, for example,as follows. RNA is prepared from an HLA-A24-restricted,MAGE-A4₁₄₃₋₁₅₁-specific CTL, for example, Clone #2-28 described inNon-Patent Publication 10 by a conventional method, and a cDNA issynthesized. 5′-Rapid Amplification of cDNA End (RACE) is performed withthe cDNA as a template, using antisense primers complementary to anucleic acid encoding a constant region of TCR α-chain and β-chain. The5′-RACE may be performed by a known method, and can be performed by, forexample, using a commercially available kit such as CapFishingFull-length cDNA Premix Kit (manufactured by Seegene Inc.). A DNAamplified by the above-mentioned technique is incorporated into aplasmid vector to transform Escherichia coli. A plasmid is produced froma transformant, and a nucleotide sequence of an inserted DNA isdetermined.

DNAs irrelevant to the TCR gene, amplified by the 5′-RACE can beexcluded by comparing the obtained nucleotide sequence to known genesequences of TCR α-chain and β-chain. In addition, there is apossibility that a DNA in which a mutation takes place in the nucleotidesequence during PCR is amplified; therefore, in the present invention,it is preferable that a sequence is determined from plural Escherichiacoli clones, and that sequences of TCR α-chain gene and β-chain geneoriginally owned by the above-mentioned CTL, that can be deduced fromthe consensus sequence are used.

The nucleic acid encoding a TCR α-chain of the amino acid sequence shownin SEQ ID NO: 1 of Sequence Listing, which has the nucleotide sequenceshown in SEQ ID NO: 3, and the nucleic acid encoding a TCR β-chain ofthe amino acid sequence shown in SEQ ID NO: 2 of Sequence Listing, whichhas the nucleotide sequence shown in SEQ ID NO: 4 are obtained by theabove-mentioned method.

In the present invention, a DNA obtained by the method mentioned abovemay be used, or a nucleic acid having the same sequence may bechemically synthesized and used.

Among the nucleic acids of the present invention, the nucleic acidencoding a moiety corresponding to a variable region of each chainconstituting the TCR can be connected to a region encoding a constantregion or intracellular region of a nucleic acid encoding otherfunctional molecules, for example, an antibody or a receptor. A novelnucleic acid thus constructed is useful in the production of a chimericfunctional molecule to be provided with an HLA-A24-restricted,MAGE-A4₁₄₃₋₁₅₁-specific binding activity.

A fourth embodiment of the present invention is a recombinant nucleicacid containing the nucleic acid of the present invention. Theabove-mentioned recombinant nucleic acid is exemplified by a nucleicacid to which various elements that enable translation of a polypeptideencoded by the above-mentioned nucleic acid are added, in a case wherethe nucleic acid of the present invention is transduced into the cell,without intending to particularly limit the present invention thereto.

The recombinant nucleic acid of the present invention consisting of aDNA is exemplified by promoters (for example, promoters derived frommammals, such as phosphoglycerate kinase promoter, Xist promoter,β-actin promoter, and RNA polymerase II promoter; and promoters derivedfrom viruses such as SV40 early promoter, cytomegalovirus promoter,thymidine kinase promoter of simple herpes virus, and LTR promoters ofvarious retroviruses), terminators, enhancers, and those having othertranscriptional controlling regions. Further, the recombinant nucleicacid may encode a sequence contributing to the translation of thepolypeptide of a first invention (Kozak sequence, and the like). It isas a matter of course that each of the above elements are arranged to afunctionally cooperative position, so as to be suitable in thetranscription of RNA and the translation of the polypeptide from thenucleic acid of the present invention. Here, in a case where therecombinant nucleic acid is RNA, an element relating to thetranscriptional control is unnecessary.

The recombinant nucleic acid of the present invention can be used byincorporating the recombinant nucleic acid into a vector as describedlater, or in the alternative, the recombinant nucleic acid can be usedin expression of the TCR by transducing the nucleic acid of the presentinvention which is an RNA directly into the cell. As the method fortransducing an RNA, a known method may be used, and for example, anelectroporation method can be suitably used.

A fifth embodiment of the present invention relates to a vectorcontaining at least one of the recombinant nucleic acids of the presentinvention, wherein the recombinant nucleic acid is inserted into thevector. The above-mentioned vector is useful in expressing anHLA-A24-restricted, MAGE-A4₁₄₃₋₁₅₁-specific TCR in a desired cell.Particularly preferred embodiments are exemplified by:

(1) a vector containing a recombinant nucleic acid containing a nucleicacid encoding a polypeptide comprising the TCR α-chain polypeptide ofthe present invention or a variable region polypeptide thereof, and arecombinant nucleic acid containing a nucleic acid encoding apolypeptide comprising the TCR β-chain polypeptide of the presentinvention or a variable region polypeptide thereof, wherein both of therecombinant nucleic acids are inserted into the vector; and(2) a combination of a vector containing a recombinant nucleic acidcontaining a nucleic acid encoding a polypeptide comprising the TCRα-chain polypeptide of the present invention or a variable regionpolypeptide thereof, wherein the recombinant nucleic acid is insertedinto the vector, and a vector comprising a recombinant nucleic acidcontaining a nucleic acid encoding a polypeptide comprising the TCRβ-chain polypeptide of the present invention or a variable regionpolypeptide thereof, wherein the recombinant nucleic acid is insertedinto the vector. In the above-mentioned (1) embodiment, the nucleic acidencoding the TCR α-chain polypeptide and the nucleic acid encoding theTCR β-chain polypeptide may be each transcribed and translated underdifferent promoters, or they may be transcribed and translated under asingle promoter by using an internal ribosome entry site (IRES).

The vector used in the present invention is not particularly limited,and a proper vector may be selected and used from a known vector such asa plasmid vector or a viral vector depending upon its purpose. Forexample, in a case where the above-mentioned recombinant nucleic acid isincorporated into a plasmid vector, in the transduction of theincorporated plasmid vector into a cell, a method of transduction, suchas calcium phosphate method, cationic lipid method, liposome method orelectroporation method can be used.

A viral vector having an ability of infecting a cell and transducing aforeign DNA is suitable in the present invention. In the presentinvention, a known viral vector, such as a retroviral vector (includinglentivirus vector and pseudo type vectors), an adenovirus vector, anadeno-associated viral vector, a herpes viral vector, can be used. Theviral vector into which the recombinant nucleic acid of the presentinvention is inserted can be infected in an intended cell underconditions appropriate for each virus, and can transduce the nucleicacid of the present invention thereinto. The retroviral vector capableof incorporating an inserted foreign nucleic acid on chromosome issuitable in the present invention.

A sixth embodiment of the present invention relates to a cell whichexpresses an HLA-A24-restricted, MAGE-A4₁₄₃₋₁₅₁-specific TCR,characterized in that the nucleic acid of the present invention istransduced into the cell. Here, the nucleic acid of the presentinvention may be transduced into a desired cell as the recombinantnucleic acid of the present invention or the vector of the presentinvention as mentioned above. A preferred embodiment of the cell of thepresent invention is exemplified by a cell into which both of a nucleicacid encoding a polypeptide comprising a TCR α-chain polypeptide or avariable region polypeptide thereof, and a nucleic acid encoding apolypeptide comprising a TCR β-chain polypeptide or a variable regionpolypeptide thereof are transduced, and a cell transformed with thevector of the present invention. Further, the present invention alsoencompasses a cell in which the above-mentioned nucleic acid isincorporated on chromosomal DNA.

Since TCR plays an important role in recognizing an antigen by a T cell,a preferred embodiment of the present invention is a T cell into whichthe nucleic acid of the present invention is transduced. A T cellexpressing an HLA-A24-restricted, MAGE-A4₁₄₃₋₁₅₁-specific TCR can beobtained by transducing the nucleic acid of the present invention into aT cell collected from a living body by the above-mentioned means.Further, as a preferred embodiment of the present invention, theabove-mentioned nucleic acid is transduced into a cell capable ofdifferentiating into a T cell, and thereafter the cell may bedifferentiated into a T cell. The cell capable of differentiating into aT cell is exemplified by, for example, hematopoietic stem cell, commonlymphoid progenitor, and a precursor cell of a T cell. Here, it is notnecessary that the cell to be transduced in which the nucleic acid istransduced is fractionated into a single cell species, and cellpopulation containing the above-mentioned cell to be transduced can be asubject for the nucleic acid transduction.

The above-mentioned cell population containing the cell to be transducedmay be collected from, for example, peripheral blood, bone marrow andcord blood of human or non-human mammal. If necessary, a T cell and/or acell capable of differentiating into a T cell can be fractionated orenriched, and used in the present invention. In a case where the TCRgene-transduced cell of the present invention is used in the treatmentof cancer or the like, it is preferable that the cell population iscollected from the patient to be treated him/herself, or a donor havingthe same HLA type as the patient.

The method of transducing the nucleic acid of the present invention intoa cell is not particularly limited, and a known method can be used. In acase where the nucleic acid of the present invention or the recombinantnucleic acid of the present invention is transduced, a method of using,for example, electroporation method, calcium phosphate method, cationiclipid method, or liposome method can be used. The nucleic acid can beconveniently and highly efficiently transduced by using a commerciallyavailable transfection reagent [for example, TransIT Series(manufactured by Mirus Bio Corporation), GeneJuice (manufactured byNovagene), RiboJuice (manufactured by Novagene), Lipofectamine(manufactured by Invitrogene)]. In a case where the vector of thepresent invention is used, the vector can be transduced into a cell inthe same manner as in the above-mentioned nucleic acid as long as thevector is a plasmid vector. On the other hand, if the vector is a viralvector, an infection method suitable for each of the vectors may beselected. Especially, in a case where a retroviral vector is used, ahighly efficient gene transfer can be performed against various cells,especially hematopoietic stem cell having a low infection efficiency ofthe retroviral vector, by using a recombinant fibronectin fragmentCH-296 (manufactured by TAKARA BIO, INC.).

A seventh embodiment of the present invention relates to a carcinostaticagent, characterized in that the carcinostatic agent contains the vectorof the fifth embodiment of the present invention or the cell of thesixth embodiment as an active ingredient. A T cell into which thenucleic acid of the present invention is transduced, obtained by thesixth embodiment of the present invention shows cytotoxic activityagainst a cell presenting an HLA-A24 molecule and MAGE-A4₁₄₃₋₁₅₁peptide. Therefore, the vector and the cell of the present inventionmentioned above can be used as a carcinostatic agent against a cancerexpressing MAGE-A4.

The above-mentioned carcinostatic agent of the present invention ischaracterized in that the carcinostatic agent contains the vector orcell of the present invention as an active ingredient. The carcinostaticagent is provided in the form in which the above-mentioned vector orcell is suspended in a pharmaceutically acceptable diluent. Here, thediluent as referred to herein is, for example, a medium suitable in thestorage of the vector or cell, physiological saline, or phosphatebuffered saline. The medium is not particularly limited, and generallyincludes media such as RPMI, AIM-V, and X-VIVO10. In addition, thecarcinostatic agent may be added with a pharmaceutically acceptablecarrier, a preservative, or the like, for the purpose of stabilization.The carrier as referred to herein is human serum albumin, and the like.The carcinostatic agent containing the cell of the present invention asan active ingredient contains the above-mentioned cell in a density ofpreferably from 1×10⁴ to 1×10⁸ cells/mL, and more preferably from 5×10⁵to 5×10⁷ cells/mL.

In a case where the carcinostatic agent containing the cell of thepresent invention as an active ingredient is administered to human, forexample, the carcinostatic agent may be administered with an injectionsyringe, and the dose per adult individual is usually so that the numberof cells mentioned above is preferably from 1×10⁶ to 1×10¹⁰ cells. Here,the above-mentioned value is a measure, and is not intended to belimited thereto. In addition, in a case of a carcinostatic agentcontaining the vector of the present invention as an active ingredient,the vector concentration and the dose in the carcinostatic agent greatlydiffer depending upon the administration route, the kinds of vectors, orthe like.

As mentioned above, according to the present invention, a method oftreating a cancer is provided. The above-mentioned method of treatmentis an in vivo gene therapy in a case where the vector of the fifthembodiment of the present invention is used as an active ingredient. Onthe other hand, in a case where the cell of the present invention isused as an active ingredient, the method is an ex vivo therapy,including the step of transducing a nucleic acid encoding anHLA-A24-restricted, MAGE-A4₁₄₃₋₁₅₁-specific TCR into a cell isolatedextracorporeally, and thereafter administering the transduced cell to apatient. The method of treatment of the present invention can use a cellderived from an individual (for example, human) to be administered, or acell derived from an individual of which HLA type is identical intowhich a nucleic acid is transduced, so that toxicity is not particularlyfound.

EXAMPLES

The present invention will be more concretely described hereinbelow bymeans of Examples, without intending to limit the present inventionthereto in any way.

Example 1 Cloning and Sequencing of TCR αChain and βChain Genes ofMAGE-A4-Specific CTL Clones #2-28

(1) Preparation of RNA from #2-28, 5′-RACE, and Cloning

#2-28 Cells, CTL clones showing cytotoxic activity to target cellspulsed with MAGE-A4₁₄₃₋₁₅₁ peptide (SEQ ID NO: 9 of Sequence Listing,hereinafter simply referred to as P143) in an HLA-A2402-restrictedmanner, described in Non-Patent Publication 10, were cultured, and RNAwas extracted from 2×10⁵ cells using RNeasy Mini Kit (manufactured byQIAGEN). cDNAs were synthesized with 200 ng of the extracted RNA as atemplate, using CapFishing Full-length cDNA Premix Kit (manufactured bySeegene, Inc.), in accordance with the instruction manual for the kit.Here, reverse transcription reaction was carried out using oligo dTadaptors as shown in SEQ ID NO: 10 of Sequence Listing, ReverseTranscriptase M-MLV (RNaseH free) (manufactured by TAKARA BIO INC.), anda buffer for the reaction appended to the above-mentioned enzyme.

PCR was carried out using the above-mentioned kit, with single-chaincDNAs thus obtained as a template. There were used 5′-RACE primersappended to the kit (SEQ ID NO: 11) as 5′ side primers and 3-TRα-Cprimers specific to TCR αchain C region (SEQ ID NO: 12), 3-TRβ-C1primers specific to TCR βchain C1 region (SEQ ID NO: 13) or 3-TRβ-C2primers specific to TCR βchain C2 region (SEQ ID NO: 14) as 3′ sideprimers. These reaction mixtures are referred to as PCR-α, PCR-β1 andPCR-β2, in that order. Each of the reaction mixtures was kept at 94° C.for 3 minutes, then repeated 30 cycles of reaction, wherein one cyclewas 94° C. for 40 seconds, 58° C. for 40 seconds and 72° C. for 1minute, and kept at 72° C. for 5 minutes. A part of each of the reactionproducts was analyzed by agarose gel electrophoresis. As a result, a DNAof about 1 kb each was amplified in PCR-α and PCR-β2.

The remaining reaction products of PCR-α and PCR-β2 were separated byagarose gel electrophoresis, and a DNA of about 1 kb each was collectedfrom the gel. The collected products were ligated with pT7 blue T-Vector(manufactured by Novagen), and Escherichia coli DH5α was transformedtherewith.

(2) Sequencing, Clustering, and Selection of Clones

Ninety-six transformants each were selected from the transformants fromPCR-α and PCR-β2 thus obtained, and plasmids were each preparedtherefrom. The DNA was sequenced using an automated sequencer. Asequence of pT7 blue was removed from the sequencing data, andthereafter subjected to clustering. As a result, the sequences of thelongest open reading frames contained in the consensus sequences of thelargest contigs were as shown in SEQ ID NO: 3 and SEQ ID NO: 4 ofSequence Listing. These sequences are cDNA sequences of TCR α chain geneand β chain gene of #2-28 cells, in turn. The amino acid sequences ofTCR α chain and β chain presumed from the cDNA nucleotide sequences areshown in SEQ ID NO: 1 and SEQ ID NO: 2 of Sequence Listing. From theabove-mentioned plasmids were selected plasmids having the consensussequences of cDNA sequences of TCR α chain gene and β chain gene in thesame direction as a T7 promoter of pT7 blue, and the plasmids were namedpBS MAGE TCR

and pBS MAGE TCRβ, respectively.

Example 2 Expression of MAGE-A4-Specific TCR According to mRNATransfection

(1) Preparation of mRNA

pBS MAGE TCR

and pBS MAGE TCRβ were linearized by digestion with EcoRI restrictionenzyme. In vitro transcription was carried out using mMESSAGE mMACHINET7 Kit (manufactured by Ambion, Inc.) with the resulting products astemplates, in accordance with the instruction manual for the kit.Thereafter, a poly(A) chain was added to the above-mentioned transcribedRNAs using Poly(A) Tailing Kit (manufactured by Ambion, Inc.) inaccordance with the instruction manual for the kit. MAGE-A4 TCR

mRNA and MAGE-A4 TCRβ mRNA were thus obtained. The resulting productswere dissolved in a phosphate buffered saline (PBS), and stored at −80°C. until use.

(2) mRNA Transfection

ms69 Cells were CTL clones showing cytotoxic activity to target cellspulsed with SAGE715-723 peptide (SEQ ID NO: 15 of Sequence Listing,hereinafter simply referred to as P715), in an HLA-A2402-restrictedmanner, but different clones from #22 obtained in the same manner as #22cells described in Non-Patent Publication 10. 1×10⁷ ms69 cells werewashed twice with X-VIVO20 medium (manufactured by Cambrex Corporation).Eighty micrograms each of MAGE-A4 TCRα mRNA and MAGE-A4 TCRβ mRNAprepared in item (1) of Example 2 were mixed with the above-mentionedcells in X-VIVO20 medium, so as to make up a total volume of 150 μL, anda transduction of the RNA into the cells was carried out according toelectroporation using ECM830 Electroporation System (manufactured byBTX). The cells into which mRNA was transduced were cultured in X-VIVO20medium at 37° C. in the presence of 5% CO₂ for one day. Hereinafter, thecells thus obtained are referred to as RNA-transduced ms69 cells.

Peripheral blood mononuclear cells (PBMCs) were separated from humanperipheral blood according to Ficoll centrifuge method, and then washedtwice with a 0.5% AB-type serum-added PBS. MACS CD8 MicroBeads(manufactured by Miltenyi Biotec KK), magnetic beads to which ananti-CD8 antibody was immobilized, were added thereto, and the mixturewas reacted at 4° C. for 15 minutes. Thereafter, the beads were washedonce with a 0.5% AB-type serum-added PBS. CD8 positive cells that werecollected from magnetic beads trapped by a column equipped with a magnetwere suspended in RPMI 1640 medium to which a 10% AB-type serum and 100U/mL interleukin 2 (IL-2) were added, so as to have a density of 1×10⁶cells/mL. An anti-CD3 antibody (Orthoclone OKT3, manufactured by JANSSENPHARMACEUTICAL K. K.) diluted with PBS so as to have a concentration of1 μg/mL was added to each well of a 24-well plate in a volume of 300μL/well each. The mixture was allowed to stand at 4° C. overnight, thesupernatant was then discarded, and the suspension of CD8 positive cellsmentioned above was put in each well in a volume of 1 mL/well each. Thecells were cultured at 37° C. in the presence of 5% CO₂ while a half thevolume of the medium was replaced on the fourth day, the seventh day andthe tenth day from the initiation of culture. 1×10⁷ CD8 positive cellson the twelfth day from the initiation of culture were washed twice withX-VIVO20 medium (manufactured by Cambrex Corporation). MAGE-A4 TCR

mRNA and MAGE-A4 TCRβ mRNA were transduced into the above-mentionedcells in the same manner as in the ms69 cells. The cells into which mRNAwas transduced were cultured in X-VIVO20 medium at 37° C. in thepresence of 5% CO₂ for one day. The cells thus obtained are hereinafterreferred to as RNA-transduced CD8 positive cells.

(3) Tetramer Assay

A polypeptide, which is HLA-A2402 heavy chain having addition of asequence acting as a substrate of biotin protein ligase BirA at aC-terminal, and β2-microglobulin were allowed to express in Escherichiacoli as an insoluble inclusion body. The above-mentioned inclusion bodywas refolded in vitro, in the presence of P143 peptide, and whereby anHLA-A2402/β2-microglobulin/P143 complex was formed. Biotin proteinligase (manufactured by Avidity Inc.) was allowed to act on theresulting complex, and a tetramer was prepared using aphycoerythrin-labeled streptavidin (Streptavidin-PE, manufactured byInvitrogen).

The above-mentioned RNA-transduced ms69 cells and RNA-transduced CD8positive cells were reacted with 20 μg/mL tetramer at 37° C. for 30minutes, and thereafter reacted with a Tricolor-labeled mouse anti-humanCD8 antibody (manufactured by CALTAG) on ice for 15 minutes. The cellswere washed, and thereafter flow cytometry analysis was carried outusing FACS Calibur (manufactured by Becton, Dickinson and Company). Thems 69 cells and the CD8 positive cells were used as negative controls,and the #2-28 cells as a positive control.

As a result, a positive percentage of the tetramer was 66.5% in theRNA-transduced ms69 cells, in contrast to 0.60% in the ms69 cells whichwere a negative control, and a positive percentage of the tetramer was34.4% in the RNA-transduced CD8 positive cells, in contrast to 23.1% inthe CD8 positive cells which were a negative control. The results oftetramer assay of the ms69 cells and CD8 positive cells into which RNAwas transduced are shown in FIG. 1 and FIG. 2. It was clarified from theresults that gene products of TCR α chain and TCR β chain cloned fromthe #2-28 cells could recognize a complex of P143 and HLA-A2402.

(4) ELISPOT Assay

Target cells were prepared as follows. T2-A24 cells prepared bytransfecting HLA-2402 gene into B×T hybrid cell line 174 CEM. T2(hereinafter simply referred to as T2 cells) (Ikuta Y. et al. Blood, 99,3717-3724 (2002)) were cultured in RPMI 1640 medium containing 10% fetalcalf serum (FCS), the cultured medium was centrifuged, and thesupernatant was then discarded. Thereafter, the cells were washed bysuspending the resulting residue in RPMI 1640 medium, centrifuging thesuspension, and then discarding the supernatant. The procedures ofwashing the cells in the manner as described above were carried out 3times in total, and thereafter the washed cells were suspended in RPMI1640 medium containing 1 mL of 10 μM P143 or P715 or without containingpeptide, and the suspension was incubated at 37° C. in the presence of5% CO₂ for 1 hour. The cells were collected by centrifugation, and thecells were then washed by suspending the collected cells in RPMI 1640medium, and centrifuging the suspension. The cells were suspended inRPMI 1640 medium so as to have a density of 5×10⁴ cells/100 μL, and thesuspension was used as the target cells of ELISPOT assay.

An anti-human interferon γ antibody (1-D1K, manufactured by Mabtech AB)diluted with PBS so as to have a concentration of 2 μg/mL was put ineach well of MultiScreen HA 96-well filter and assay plate (manufacturedby Millipore Corporation) in a volume of 100 μL/well each, and allowedto stand at 4° C. overnight. The liquid in the wells was discarded, andthereafter the plate was washed by adding RPMI 1640 medium to each wellin a volume of 100 μL/well each, allowing the mixture to stand for 15minutes, and discarding the liquid. The washing procedures were carriedout one more time, and thereafter RPMI 1640 medium containing 10%AB-type serum was added to each well, and the mixture was allowed tostand at 37° C. for 1 hour to carry out blocking. After the blocking,the supernatant was aspirated, and thereafter the plate was washed byadding RPMI 1640 medium to each well in a volume of 100 μL each. Thewashing procedures were carried out 3 times in total.

(a) The RNA-transduced ms69 cells, (b) the ms69 cells which were anegative control and (c) the #2-28 cells which were a positive control,each prepared in item (2) of Example 2, and (d) the RNA-transduced CD8positive cells and (e) the CD8 positive cells which were a negativecontrol, each prepared in item (2) of Example 2 were collected bycentrifugation, and the collected cells were once washed with RPMI 1640medium. The cells (a), (b) and (c) were suspended in RPMI 1640 medium,so as to have a density of 2000 cells/100 μL, 1000 cells/100 μL or 500cells/100 μL, and the cells (d) and (e) were suspended so as to have adensity of 2×10⁴ cells/100 μL, and the suspension was put in the wellsof washed plate prepared in item (4) of Example 2 in a volume of 100μL/well each. The suspension of target cells mentioned above was addedthereto in a volume of 100 μL each, and the mixture was cultured at 37°C. in the presence of 5% CO₂ for 20 hours.

The liquid was removed from each well of the plate and each well waswashed 6 times with PBS containing 0.05% Tween 20 (PBS-T). Biotinylatedanti-human interferon γ antibodies (manufactured by Mabtech AB, a clonename of 7-B6-1) were diluted with PBS so as to have a concentration of0.2 μg/mL, and the dilution was put in each well in a volume of 100 μLeach, and thereafter allowed to stand at 4° C. overnight.

The liquid was removed from each well of the plate, and each well waswashed 6 times with PBS-T. An alkaline phosphatase-labeled streptavidin(manufactured by Bio-Rad Japan) diluted with PBS so as to have aconcentration of 1 μg/mL was put in each well in a volume of 100 μLeach, and the mixture was reacted at room temperature for 1 hour. Theliquid was removed from each well of the plate, and each well was washed3 times with PBS-T. Thereafter, a color development solution prepared inaccordance with the instruction manual for AP Color Development Kit(manufactured by Bio-Rad Japan, 170-6432) was put in each well in avolume of 100 μL each, and a color development reaction was carried outwith shading. The color development was stopped with distilled water,and a photograph of the plate was taken.

As a result, the ms69 cells and CD8 positive cells into which RNA wastransduced formed a larger number of interferon γ positive spots whenthe target cells were pulsed with P143, as compared to those of the ms69cells and the CD8 positive cells. The results are shown in FIG. 3 andFIG. 4. FIG. 3 is the results of ELISPOT assay when the CTL clones wereused as effector cells. A large number of interferon γ positive spotswere formed by the ms69 cells to the target cells pulsed with P715, the#2-28 cells to the target cells pulsed with P143, and the RNA-transducedms69 cells to both target cells. FIG. 4 is the results of ELISPOT assaywhen the CD8 positive cells were used as effector cells. Interferon γpositive spots were formed by the RNA-transduced CD8 positive cells,specific to the target cells pulsed with P143.

(5) Cytotoxic Activity

The T2 cells, the T2-A24 cells, and T2-A2 cells prepared by transfectingHLA-A0201 gene into T2 cells were washed 3 times with RPMI 1640 medium,and the washed cells were suspended in RPMI 1640 medium so as to have adensity of 5×10⁶ cells/mL. P143 or P715 was added to 1 mL each of theabove-mentioned cell suspensions so as to have a final concentration of10 μM, and the mixture was allowed to stand at room temperature for 15minutes. Thereafter, 1 mL each of RPMI 1640 medium containing 10% FCSwas added thereto, and the mixture was incubated at 37° C. for 1 hour.The cells were washed 3 times with RPMI 1640 medium without containingFCS, and 1×10⁶ cells were suspended in 100 μL of RPMI 1640 mediumcontaining 10% FCS. Fifty microliters of an aqueous Na₂ ⁵¹CrO₄ solution(3.7 MBq) was added thereto, and the mixture was labeled at 37° C. for 2hours. The resulting cells were used as the target cells for thedetermination of cytotoxic activity.

The ms69 cells, the RNA-transduced ms69 cells, the #2-28 cells, the CD8positive cells, and the RNA-transduced CD8 cells were washed twice withRPMI 1640 medium, and the washed cells were suspended in RPMI 1640medium containing 10% FCS so as to have a density of 2×10⁶ cells/mL,1×10⁶ cells/mL, 5×10⁵ cells/mL, 2.5×10⁵ cells/mL, 1.25×10⁵ cells/mL, and6.25×10⁴ cells/mL (effector cells), respectively, and a 100 μL portionthereof was put in each well of a 96-well V-bottom plate. The targetcells were suspended in RPMI 1640 medium containing 10% FCS so as tohave a density of 1×10⁶ cells/mL, and the suspension was added to eachwell with the effector cells in a volume of 100 μL each. The mixture wasreacted at 37° C. for 4 hours, and thereafter the supernatants werecollected by centrifugation, and an amount of ⁵¹Cr released to 100 μL ofthe supernatant was determined by using a gamma counter. The specificcytotoxic activity was calculated from the measured value ofradioactivity according to the following formula.Specific Cytotoxic Activity(%)=[(Measured Value in Each Well−MinimumRelease Value)/(Maximum Release Value−Minimum ReleaseValue)]×100  [Formula 1]

In the above-mentioned formula, the minimum release value is the amountof ⁵¹Cr released in the well to which the effector cells are not added,showing the amount of ⁵¹Cr naturally released from the target cells. Inaddition, the maximum release value shows the amount of ⁵¹Cr releasedwhen the cells are disrupted by adding Triton X-100 to the target cells.

The results are shown in FIG. 5 and FIG. 6. FIG. 5 is graphs showingcytotoxic activity of CTL clones to (a) T2-A24 cells pulsed with P715,(b) T2-A24 cells pulsed with P143, and (c) T2-A2 cells pulsed with P143,and the axis of abscissas shows a ratio of the number of effectorcells/the number of target cells (E/T ratio), and the axis of ordinatesshows specific cytotoxic activity (%). The ms69 cells showed cytotoxicactivity only to the T2-A24 cells pulsed with P715, and the #2-28 cellsshowed cytotoxic activity only to the T2-A24 cells pulsed with P143; incontrast, the ms69 cells into which RNA was transduced showed cytotoxicactivity to both target cells. Any of the effector cells did not showcytotoxic activity to the T2-A2 cells pulsed with P143. FIG. 6 is graphsshowing cytotoxic activity of CD8 cells to (a) T2 cells pulsed withP143, (b) T2-A24 cells which were not pulsed with peptide, and (c)T2-A24 cells pulsed with P143, and the axis of abscissas shows the E/Tratio, and the axis of ordinates shows specific cytotoxic activity (%).The CD8 positive cells into which RNA was transduced and the #2-28 cellswhich are a positive control showed cytotoxic activity to the T2-A24cells pulsed with P143, while the CD8 positive cells which are anegative control did not show cytotoxic activity to any of the cells.

It was clarified from the above results that the gene encoding TCR αchain and β chain of #2-28 cells gives a P143-specificHLA-A2402-restricted cytotoxic activity to the CTL clones and the CD8cells derived from peripheral blood.

INDUSTRIAL APPLICABILITY

According to the present invention, a HLA-A24-restricted, TCR α chainand β chain polypeptide to MAGE-A4, derived from CTL, and a nucleic acidencoding the polypeptide are provided. Since the above-mentioned nucleicacid can give T cells cytotoxic activity to a cell presenting HLA-A24molecule and MAGE-A4₁₄₃₋₁₅₁ peptide, the above-mentioned nucleic acid isuseful in the treatment of a cancer that expresses MAGE-A4.

SEQUENCE LISTING FREE TEXT

SEQ ID NO: 10; Oligo dT adaptor.

SEQ ID NO: 11; 5′-RACE primer.

SEQ ID NO: 12; Synthetic primer 3-TRalpha-C to amplify a DNA fragmentencoding TCR alpha chain.

SEQ ID NO: 13; Synthetic primer 3-TRbeta-C1 to amplify a DNA fragmentencoding TCR beta chain.

SEQ ID NO: 14; Synthetic primer 3-TRbeta-C2 to amplify a DNA fragmentencoding TCR beta chain.

1. A recombinant nucleic acid encoding a polypeptide comprising theamino acid sequence of SEQ ID NO:
 5. 2. The recombinant nucleic acidaccording to claim 1, wherein the nucleic acid comprises the nucleotidesequence of SEQ ID NO:
 6. 3. A recombinant nucleic acid encoding apolypeptide comprising the amino acid sequence of SEQ ID NO:
 7. 4. Therecombinant nucleic acid according to claim 3, wherein the nucleic acidcomprises the nucleotide sequence shown in SEQ ID NO:
 8. 5. Therecombinant nucleic acid according to claim 1, wherein the nucleic acidencodes a polypeptide comprising the amino acid sequence of SEQ IDNO:
 1. 6. The recombinant nucleic acid according to claim 2, wherein thenucleic acid comprises the nucleotide sequence of SEQ ID NO:
 3. 7. Therecombinant nucleic acid according to claim 3, wherein the nucleic acidencodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2.
 8. The recombinant nucleic acid according to claim 4, wherein thenucleic acid is a nucleic acid comprising the nucleotide sequence of SEQID NO:
 4. 9. The recombinant nucleic acid according to claim 1, whereinthe nucleic acid encodes a polypeptide consisting of the amino acidsequence of SEQ ID NO: 1 or SEQ ID NO:
 5. 10. The recombinant nucleicacid according to claim 2, wherein the nucleic acid consists of thenucleotide sequence of SEQ ID NO: 3 or SEQ ID NO:
 6. 11. The recombinantnucleic acid according to claim 3, wherein the nucleic acid encodes apolypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or SEQID NO:
 7. 12. The recombinant nucleic acid according to claim 4, whereinthe nucleic acid is a nucleic acid consisting of the nucleotide sequenceof SEQ ID NO: 4 or SEQ ID NO:
 8. 13. A vector comprising the recombinantnucleic acid as defined in claim 1 or claim 5, wherein the recombinantnucleic acid is inserted into the vector.
 14. A vector selected from thegroup consisting of: (1) a vector comprising a recombinant nucleic acidcomprising the nucleic acid as defined in claim 1 or claim 5, and arecombinant nucleic acid comprising the nucleic acid as defined in claim3 or claim 7, wherein both of the recombinant nucleic acids are insertedinto the vector; and (2) a combination of vectors comprising arecombinant nucleic acid comprising the nucleic acid as defined in claim1 or claim 5, wherein the recombinant nucleic acid is inserted into afirst vector, and a second vector comprising a recombinant nucleic acidcomprising the nucleic acid as defined in claim 3 or claim 7, whereinthe recombinant nucleic acid is inserted into the second vector.
 15. Acarcinostatic agent comprising an active ingredient selected from thegroup consisting of: (1) a vector comprising the recombinant nucleicacid as defined in claim 1 or claim 5, wherein the recombinant nucleicacid is inserted into the vector, (2) a vector comprising therecombinant nucleic acid as defined in claim 3 or claim 7, wherein therecombinant nucleic acid is inserted into the vector, (3) a vectorcomprising a recombinant nucleic acid comprising the nucleic acid asdefined in claim 1 or claim 5, and a recombinant nucleic acid comprisingthe nucleic acid as defined in claim 3 or claim 7, wherein both of therecombinant nucleic acids are inserted into the vector; and (4) acombination of vectors comprising a recombinant nucleic acid comprisingthe nucleic acid as defined in claim 1 or claim 5, wherein therecombinant nucleic acid is inserted into a first vector, and a secondvector comprising a recombinant nucleic acid comprising the nucleic acidas defined in claim 3 or claim 7, wherein the recombinant nucleic acidis inserted into the second vector.
 16. A vector comprising therecombinant nucleic acid as defined in claim 3 or claim 7.